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Toxicological assessment of biodegraded pentachlorophenol: Microtox and fish embryos.

A Gram-negative bacterium, Pseudomonas sp. strain SR3, was isolated from soil at a former wood treatment plant in north central Florida. The ability of this bacterium to degrade pentachlorophenol (PCP) was confirmed by growing cells in a basal salts medium in which PCP was the only source of carbon and energy. Degradation from a measured concentration of 39-40 micrograms PCP/ml to 0.0006 micrograms PCP/ml was observed within 120 h of incubation in the presence of PCP-induced cells of Pseudomonas sp. strain SR3. The initial cell density in these cultures was 6 x 10(6) cfu/ml. Microtox 5 min EC50 toxicity tests revealed that aqueous solutions of PCP, measured concentrations 39-40 micrograms/ml were toxic but that final biodegraded samples, 0.0006 micrograms PCP/ml were nontoxic. However, bioassays with embryonic inland silversides, Menidia beryllina, showed that the biodegraded samples were embryotoxic or teratogenic. Water containing added PCP at concentrations up to 30 times higher than measured in the final biodegraded samples was less toxic/teratogenic. These results indicate that while biodegradation of PCP was nearly complete, intermediate metabolites of the degradation process or undegraded impurities in PCP were toxic or teratogenic. Thus, the M. beryllina bioassay allows extremely sensitive assessment of toxicity associated with biodegraded environmental pollutants and may be a useful criterion for determining whether bioremediated water or soil is safe for discharge back into the environment.

Abnormalities, Drug-Induced↗

Residues of pentachlorophenol and other chlorinated contaminants in human tissues: analysis by electron capture gas chromatography and electron capture negative ion mass spectrometry.

Samples of human tissues including testes, kidneys, prostate glands, livers, and adipose tissues removed at autopsy were analyzed for pentachlorophenol (PCP) and nonachloro-2-phenoxyphenol (NCPP); the fat samples were also analyzed for other chlorinated contaminants. Electron capture gas chromatography was used to quantitate the residues after isolation and cleanup. Identity of the residues was confirmed by electron capture negative ion mass spectrometry. All tissues analyzed for PCP tested positive with a range from 0.007 ppm (microgram/g) in subcutaneous fat to 4.14 ppm in testis. Residues of NCPP were much lower, ranging from levels below the detection limit to 0.59 ppm in testis. The highest average residues, based on the lipid content of the tissue, of PCP (1.09 ppm) and of NCPP (0.19 ppm) were found in testis followed by kidney (0.95 ppm PCP), prostate (0.84 ppm PCP), and liver (0.59 ppm PCP). Residues of these chemicals in the adipose tissues were on the average about 40 times lower than the residues in the non-fatty tissues. Other chlorinated contaminants found in the subcutaneous fat included hexa-, hepta-, and octa-chlorodibenzo-p-dioxins at sub-ppb levels and DDE averaging 2.47 ppm.

Adipose Tissue↗

Assessment of inhibition kinetics of the growth of strain P5 on pentachlorophenol under steady-state conditions in a nutristat.

A bacterium degrading pentachlorophenol (PCP) as the only source of carbon and energy was grown in a nutristat , i.e., a continuous culture with on-line measurement and control of the substrate concentration. We improved the PCP nutristat by incorporation of a personal computer with a proportional integral derivative (PID) algorithm for controlling the medium feed pump. The controlled value deviated from the average (set-point) value by 1% maximally. In the PCP nutristat (30 degrees C), the steady-state dilution rate, and hence, specific growth rate, showed a maximum value of 0.142 +/- 0.004 h-1 at set-point PCP concentrations between 37 and 168 microM. At PCP concentrations above 168 microM, the steady-state growth rate decreased because of inhibition. The growth yield coefficient was not seriously affected by the PCP concentration, suggesting that uncoupling was not the inhibitory mechanism. It was concluded that the PCP nutristat is very useful for establishing steady-state conditions that maintain growth-inhibitory PCP concentrations and high cell concentrations, conditions for which the chemostat is not suitable.

Bacteriological Techniques↗

Suppression of mouse complement activity by contaminants of technical grade pentachlorophenol.

Pentachlorophenol (PCP) is an antimicrobial agent used chiefly for the preservation of wood. Subchronic oral exposure (14 days) to Technical Grade PCP significantly inhibited the functional activity of female B6C3F1 mouse complement when measured in a microtiter hemolytic assay. When evaluated one day following the final exposure the highest administered dose (100 mg/kg) significantly suppressed the Classical complement pathway, the Spontaneous C1 autoactivation pathway, the Alternate pathway and the level of complement component, C3. Reconstitution studies using C5-deficient serum also demonstrated deleterious effects on this complement component. The Classical pathway was the most sensitive to Technical Grade PCP effects. Animals treated with 100 mg/kg Technical Grade PCP had CH 50 levels 30% of vehicle controls. Animals treated for 14 days and allowed a 15 day recovery period had CH 50 values 36% of control and animals which recovered for 30 days had only 52% of the complement activity of control animals. C3 recovery studies also demonstrated continued suppression on days 15 and 30 post-final exposure. Doses of 10 and 30 mg/kg did not produce the marked effects observed with the highest dose; however, a dose-dependent trend was observed for all responses. Animals treated with 100 mg/kg of EC-7, a PCP preparation with reduced amounts of contaminating dioxins and dibenzofurans, did not demonstrate detrimental effects on the complement system.

Animals↗

Use of a pentachlorophenol degrading bacterium to bioremediate highly contaminated soil.

A Sphingomonas species that mineralizes high concentrations of pentachlorophenol (PCP) was isolated from a PCP-contaminated EPA Superfund site. This bacterium, identified as Sphingomonas sp. strain RA2, is able to degrade PCP at concentrations of up to 300 micrograms/mL in liquid culture. This organism was tested for its ability to degrade high concentrations of PCP in a soil that did not contain organisms capable of degrading high concentrations of PCP. When inoculated into contaminated soil, Sphingomonas sp. RA2 mineralized PCP at concentrations of 300, 600, 900, and 1200 micrograms PCP/g of soil, but was unable to mineralize 1500 micrograms PCP/g of soil. Only very minimal loss of PCP was seen in uninoculated soils. The results of this study demonstrate that Sphingomonas sp. RA2 may be a useful organism for remediation of sites contaminated with high concentrations of PCP.

Biodegradation, Environmental↗

Fractionation of protein adducts in rats and mice dosed with [14C]pentachlorophenol.

Pentachlorophenol (PCP) induces liver cancer in mice, possibly due to covalent binding of PCP metabolites to critical macromolecules. In this work, covalent binding was related to PCP biotransformation and specific (cysteinyl) adducts of chlorinated quinones in liver and blood of Sprague-Dawley rats and B6C3F1 mice dosed with [(14)C]PCP. Using a sequential scheme of scintillation counting along with selective cleavage of cysteinyl adducts by Raney nickel, we quantified total radiobinding, total covalent binding, non-cysteinyl protein binding, and specific protein adducts in liver nuclei (Np), liver cytosol (Cp), hemoglobin (Hb), and serum albumin (Alb). Almost all of the radiobinding to Np (>98%) was attributed to covalent binding in both rats and mice. Regarding Cp, more covalent binding was observed in mice than in rats (100% versus 67%, P=0.015). Very little binding was attributed to serum Alb (rats 1.3%, mice 2.6%, P=0.046) or Hb (not detected in either species). These results indicate that the liver was the main organ for PCP metabolism and that relatively little of the dose of reactive metabolites became systemically available. Cysteinyl binding accounted for 76-91% of total covalent binding to Np and 68-76% of total covalent binding to Cp. In addition, five times more PCP was bioactivated in the livers of mice than in those of rats (2.14% of the dose bound to Cp in mice and 0.416% in rats). These results reinforce previous studies, suggesting that the liver was a target organ of PCP carcinogenicity and that mice were more susceptible to liver damage than rats. However, the sum of all quantified adducts accounted for only 7-8% of total cysteinyl binding to Np and 2% to Cp, suggesting that other uncharacterized binding species may be important to the toxicity of PCP.

Animals↗

Oxidative DNA lesions in V79 cells mediated by pentachlorophenol metabolites.

Incubation of the pentachlorophenol (PCP) metabolites, tetrachloro-p-benzoquinone (chloranil, TCpBQ), tetrachloro-p-hydroquinone (TCpHQ) and tetrachloro-p-benzoquinone (TCoBQ) with V79 Chinese hamster cells led to a significant enhancement of the amount of 8-hydroxydeoxyguanosine (8-OH-dG) in DNA. With PCP itself and the metabolite tetrachloro-o-hydroquinone (TCoHQ) no distinct induction of this lesion could be observed. The average yields of 8-OH-dG were about 2-2.5 times above background levels. In addition, TCpBQ and TCpHQ were able to generate DNA single-strand breaks, while PCP, TCoHQ and TCoBQ failed to induce this lesion. All incubations were performed for 1 h without exogenous metabolic activation and concentrations were 25 microM of the respective agent. It is concluded that these metabolites may contribute to the carcinogenicity of PCP observed in mice, by generating reactive oxygen species (ROS) through their redox cycling properties.

8-Hydroxy-2'-Deoxyguanosine↗

Mixture component effects on the in vitro dermal absorption of pentachlorophenol.

Interactions between chemicals in a mixture and interactions of mixture components with the skin can significantly alter the rate and extent of percutaneous absorption, as well as the cutaneous disposition of a topically applied chemical. The predictive ability of dermal absorption models, and consequently the dermal risk assessment process, would be greatly improved by the elucidation and characterization of these interactions. Pentachlorophenol (PCP), a compound known to penetrate the skin readily, was used as a marker compound to examine mixture component effects using in vitro porcine skin models. PCP was administered in ethanol or in a 40% ethanol/60% water mixture or a 40% ethanol/60% water mixture containing either the rubefacient methyl nicotinate (MNA) or the surfactant sodium lauryl sulfate (SLS), or both MNA and SLS. Experiments were also conducted with 14C-labelled 3,3',4,4'-tetrachlorobiphenyl (TCB) and 3,3',4,4',5-pentachlorobiphenyl (PCB). Maximal PCP absorption was 14.12% of the applied dose from the mixture containing SLS, MNA, ethanol and water. However, when PCP was administered in ethanol only, absorption was only 1.12% of the applied dose. There were also qualitative differences among the absorption profiles for the different PCP mixtures. In contrast with the PCP results, absorption of TCB or PCB was negligible in perfused porcine skin, with only 0.14% of the applied TCB dose and 0.05% of the applied PCB dose being maximally absorbed. The low absorption levels for the PCB congeners precluded the identification of mixture component effects. These results suggest that dermal absorption estimates from a single chemical exposure may not reflect absorption seen after exposure as a chemical mixture and that absorption of both TCB and PCB are minimal in this model system.

Administration, Topical↗

Microwave-assisted steam distillation with simultaneous liquid/liquid extraction of pentachlorophenol from organic wastes and soils.

An efficient method for extracting pentachlorophenol (PCP) from organic solid matrices and soils using direct excitation of the fresh samples by microwave energy is presented. Steam-volatile PCP partitioned into the organic solvent n-hexane within the closed extraction vessel, resulting in recovery rates of 85-92% from aged samples. Condensed water provided a boundary layer between extract and sample thus preventing any contact of PCP with and re-partitioning into the sample. Duration of the microwave-assisted process was 35 min. Crude extracts were derivatized by heptafluorobutyric anhydride (HFBA) to improve selectivity and sensitivity resulting in detection limits of 1-2 micro g kg(-1). GC-MS/MS analyses proved that suitable extracts with only minor impurities were obtained. Common sample treatment steps like drying, thorough grinding, frequent transferring, and tedious clean-up, and concentration procedures which all can cause certain losses of analyte were minimized. The efficiency of the method was verified by comparison with an established ultrasonic extraction procedure. This microwave-assisted pressurized steam distillation with simultaneous partition into an organic phase thus provides a streamlined and efficient strategy which requires no additional investment in standard equipment for microwave-assisted extractions (MAE). Degradation of analytes at longer extraction times must be taken into account.

Chemical Fractionation↗

Assessing contaminant sensitivity of endangered and threatened aquatic species: part II. Chronic toxicity of copper and pentachlorophenol to two endangered species and two surrogate species.

Early life-stage toxicity tests with copper and pentachlorophenol (PCP) were conducted with two species listed under the United States Endangered Species Act (the endangered fountain darter, Etheostoma fonticola, and the threatened spotfin chub, Cyprinella monacha) and two commonly tested species (fathead minnow, Pimephales promelas, and rainbow trout, Oncorhynchus mykiss). Results were compared using lowest-observed effect concentrations (LOECs) based on statistical hypothesis tests and by point estimates derived by linear interpolation and logistic regression. Sublethal end points, growth (mean individual dry weight) and biomass (total dry weight per replicate) were usually more sensitive than survival. The biomass end point was equally sensitive as growth and had less among-test variation. Effect concentrations based on linear interpolation were less variable than LOECs, which corresponded to effects ranging from 9% to 76% relative to controls and were consistent with thresholds based on logistic regression. Fountain darter was the most sensitive species for both chemicals tested, with effect concentrations for biomass at < or = 11 microg/L (LOEC and 25% inhibition concentration [IC25]) for copper and at 21 microg/L (IC25) for PCP, but spotfin chub was no more sensitive than the commonly tested species. Effect concentrations for fountain darter were lower than current chronic water quality criteria for both copper and PCP. Protectiveness of chronic water-quality criteria for threatened and endangered species could be improved by the use of safety factors or by conducting additional chronic toxicity tests with species and chemicals of concern.

Animals↗

Humic acid toxicity in biologically treated soil contaminated with polycyclic aromatic hydrocarbons and pentachlorophenol.

Contaminated soil from a land treatment unit at the Libby Groundwater Superfund Site in Libby, MT, was amended with 14C pyrene and incubated for 396 days to promote biodegradation and the formation of soil-associated bound residues. Humic and fulvic acids were extracted from the treated soil microcosms and analyzed for the presence of pyrene residues. Biologic activity promoted 14C association with the fulvic acid fraction, but humic acid-associated 14C did not increase with biologic activity. The Aboatox flash toxicity assay was used to assess the toxicity of humic and fulvic acid fractions. The fulvic acid gave no toxic response, but the humic acid showed significant toxicity. The observed toxicity was likely associated with pentachlorophenol, a known contaminant of the soil that was removed by solvent extraction of the humic acid and that correlated well with toxicity reduction.

Aliivibrio fischeri↗

Development of stable isotope dilution quantification liquid chromatography-mass spectrometry method for estimation of exposure levels of bisphenol A, 4-tert-octylphenol, 4-nonylphenol, tetrabromobisphenol A, and pentachlorophenol in indoor air.

The potential risks to human health and reproduction of phenolic xenoestrogens, such as bisphenol A (BPA), 4-tert-octylphenol (OP), 4-nonylphenol (NP), tetrabromobisphenol A (TBBPA), and pentachlorophenol (PCP), have not been fully elucidated. It is possible that healthy humans are exposed to phenolic xenoestrogens in their daily lives. It is also possible that the source of human exposure to trace amounts of phenolic xenoestrogens is indoor air. Therefore, we have established an accurate, sensitive, and selective method for determining BPA, OP, NP, TBBPA, and PCP in air samples by using stable isotope dilution techniques using 13C12-BPA, 4-(1-methyl) octylphenol-d5 (m-OP-d5), 13C12-TBBPA, 13C6-PCP, and liquid chromatography-mass spectrometry (LC-MS). Air sampling (71/min, 24 hours), using glass filters (GB-100R, 47 mm) and Empore SDB-XD filters (47 mm/0.5 mm, 3M, Tokyo, Japan) and subsequent extraction by washing were carried out. Then the extracted sample solution was subjected to LC-MS. The percentage recovery of these analytes ranged from 87.0% to 101.9% (SD 0.2% to 4.6%, n=3) with correction using the stable isotope dilution quantification method. The quantification limit (signal-to-noise ratio, S/N>10) in the air samples was 0.1 ng/m3. Our findings suggest that the LC-MS method can detect low levels of phenolic xenoestrogens in air samples and that these trace levels of analytes exist in indoor air.

Air Pollution, Indoor↗

Uptake and elimination of chlorpyrifos and pentachlorophenol into the freshwater amphipod Gammarus pulex.

Uptake and elimination rates were determined for chlorpyrifos (CPF) and pentachlorophenol (PCP) in the freshwater amphipod Gammarus pulex. Internal concentrations of the two pesticides were measured over a three-day exposure phase and a subsequent three-day elimination phase. Rate constants were obtained by fitting measured internal concentrations to a one-compartment single first-order model. The uptake rate constants were 747+/-61 [L kg-1 day-1] for CPF and 89+/-7 [L kg-1 day-1] for PCP. The elimination rate constants were 0.45+/-0.05 [day-1] for CPF and 1.76+/-0.14 [day-1] for PCP. The resulting bioconcentration factors at steady state were 1660 and 51 for CPF and PCP, respectively. The parameter estimation method and possible variability due to varying lipid content are briefly discussed.

Amphipoda↗

Sorption of the neutral and charged forms of pentachlorophenol on soil: evidence for different mechanisms.

Laboratory soil sorption experiments have been conducted on pentachlorophenol (PCP) at different pH values in an attempt to elucidate differences in sorption mechanisms between the charged and neutral species. Sorption of PCP on soil was investigated by maintaining pH 4 or 8 in batch sorption experiments. Pre-equilibration of the soil was necessary to maintain a constant pH over the course of the experiments. Additionally, a CaCO3-CO2/N2 buffered solution was necessary to maintain a pH of 8. Sorption of the neutral PCP species conformed to a linear isotherm model, while a Langmuir model provided the best fit for the charged species. Desorption of the neutral form was completely reversible over the sorption times studied but the charged species exhibited some resistance to desorption. Temperature effects on the distribution coefficients (Kd) were investigated and thermodynamic parameters were calculated. The ionized species showed a clear decrease in Kd with increasing temperature while the protonated species showed no apparent trend. Enthalpies (deltaHo), entropies (deltaSo), and free energies (deltaGo), support the conclusions that the neutral form of PCP partitions by hydrophobically binding to the soil while the charged form sorbs by a more specific exothermic adsorption reaction.

Adsorption↗

Comparative fate of 1,1-diphenylethylene (DPE), 1,1-dichloro-2,2-bis(4-Chlorophenyl)-ethylene (DDE), and pentachlorophenol (PCP) under alternating aerobic and anaerobic conditions.

Bacterial degradation of 1,1-dichloro-2,2-bis-(4-chlorophenyl)-ethylene (DDE) and its dehalogenated derivative 1,1-diphenylethylene (DPE) has not yet been shown and may require culture adaptation and special culture conditions. We compared the degradability of DPE, DDE, and pentachlorophenol (PCP) in aerobic/anaerobic sequenced batch reactor systems. Reactors operated under aerobic/methanogenic and aerobic/denitrifying conditions were inoculated with bacterial consortia from anaerobic granular sludge, long-term PCP- and DDE-contaminated soil, and pulp and paper waste pond sediment. The culture was gradually acclimatized to low concentrations of DPE, DDE, and PCP in defined minimal growth media with benzoate, phenol, ethanol, and formate as primary carbon sources. DDE remained refractory for 105 days, whereas DPE and PCP were degraded. This suggests that DDE is extremely recalcitrant to degradation by aromatic organochlorine-degrading bacteria from long-term polluted soils and sediments. The results confirm that the chlorination of DDE is a major biodegradation barrier for adapted bacteria under aerobic and anaerobic conditions.

Bacteria, Aerobic↗

A novel catabolic activity of Pseudomonas veronii in biotransformation of pentachlorophenol.

Pseudomonas veronii PH-05, a bacterial strain capable of transforming pentachlorophenol (PCP) to a metabolic intermediate, was isolated by selective enrichment of soil samples from a timber storage yard. Strain PH-05 was shown to be able to grow using PCP as the sole source of carbon and energy. GC-MS analysis showed that the metabolic intermediate was tetrachlorocatechol, which inhibited the growth of this strain. The formation of tetrachlorocatechol during biotransformation was monitored, and its inhibitory effect on growth of strain PH-05 was analyzed at a range of concentrations. The catabolic activity of the isolated strain differs from that of other PCP-degrading bacteria, which metabolize PCP through a chlorinated hydroquinone intermediate.

Biotransformation↗

Bioavailability of pentachlorophenol to acclimatised bacteria under batch and flow-through conditions.

Biodegradation of organic contaminants in multi-phase systems, such as soils and aquifers, is often limited by the extent and the rate of contaminant sorption onto the solid matrix. However, information about biodegradation in complex matrices is largely limited to studies of closed systems under batch conditions in which potential bioavailability is characterised by the aqueous-phase concentration of a contaminant. There is little knowledge regarding the influence of flow-through conditions on the availability of contaminants to microbes (contaminant bioavailability). Thus, the aim of this study was to assess and compare contaminant bioavailability, in the presence of a sorptive medium, under both batch and flow-through conditions. Accordingly, experiments were designed in which pentachlorophenol (PCP) was introduced into a mixture of inoculated silica sand and a PCP-retaining resin, under either batch or flow-through (columns) conditions. The results indicated that an increase in the amount of resin (0.1-0.2 g) clearly lowered PCP availability to microbes after 170 h under batch conditions (30 and 45% respectively); whereas, the initial decrease in bioavailability observed under flow-through conditions (45 and 70% respectively) was reversed and no longer observable after 170 h. This increase in PCP availability was linked to an improvement in the contaminant biodegradation capacity from 0.03 to 0.13 mg x l(-1) x h(-1) over 200 h.

Adsorption↗